Crystallization conditions and treatments.

<p>Crystallization conditions and treatments.</p

Dimerization is possibly mediated by the N-terminal domain.

<p>(a) SEC/MALS indicates monomer-dimer equilibrium for full-length CNPase. The calculated molecular masses (black) for the two peaks are 78 and 44 kDa, based on light scattering and refractive index. Higher-order oligomers are also detectable. (b) Analysis of the purified N-terminal domain by SEC indicates the presence of dimers. The elution volumes of molecular weight standards run on the same column are indicated above the graph (in kDa). The expected monomeric molecular weight is 19 kDa, and the elution volume is compatible with the presence of a slightly elongated dimer.</p

The overall structure.

<p>(a) Crystal structure of the mouse CNPase phosphodiesterase domain. The side chains of the conserved HxTx motifs and the bound 2′-AMP in the active site are shown as sticks. The protein is colored from blue (N-terminus) to red (C-terminus), and secondary structure elements are labelled. (b) Superposition of the three different crystal forms of mouse CNPase determined in this study, indicating flexibility mainly in 4 loops.</p

RNA binding.

<p>(a) Elution profiles of purified full-length CNPase with (blue) and without (red) RNase A treatment. (b) Gel electrophoresis of fractions from size exclusion chromatography. Top: SDS-PAGE analysis of the void volume (1) and the monomer peak (2). Middle: The same samples run on an agarose gel with ethidium bromide staining. Bottom: A sample from the void volume with (+) and without (−) RNase treatment confirms the presence of RNA. (c) Binding of different CNPase constructs to a polyadenylyl affinity matrix. 1 – input sample; 2–6 – washes; 7 – bound protein eluted from the matrix with sample buffer. FL – full-length; C – catalytic domain; N – N-terminal domain. (d) The potential beginning of the RNA-binding site in CNPase. The large hydrophobic surface extends from the active site towards the N-terminal domain. The hydrophobic region is surrounded by a rim of positive electrostatic potential. The colouring is from −0.3 V (red) to +0.3 V (blue), as implemented in ccp4mg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032336#pone.0032336-McNicholas1" target="_blank">[51]</a>. The magenta arrow indicates the suggested binding surface for RNA. The N- and C-termini for the domain are labeled. (e) Stereo view of the aromatic cluster in the small lobe.</p

Conformation of the CNPase catalytic domain as detected by SAXS.

<p>(a) Left: Experimental scattering curve for the CNPase catalytic domain (red dots) overlaid with the theoretical scattering curve computed using the crystal structure (black line). The Guinier plot corresponding to this scattering curve is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032336#pone.0032336.s004" target="_blank">Figure S4</a>. Middle: Distance distribution function. Right: Averaged <i>ab initio</i> model of the phosphodiesterase domain (white surface) superimposed with the crystal structure (magenta). (b) SAXS analysis of extended CNPase catalytic domain constructs – left: scattering curves, middle: distance distribution functions. Black: the crystallized catalytic domain; red: construct cat-C; blue: construct N-cat; green: construct N-cat-C. The scattering curves are slightly displaced in the y direction for easier viewing: the corresponding Guinier plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032336#pone.0032336.s004" target="_blank">Figure S4</a>. Right: <i>Ab initio</i> models of the catalytic domain (gray), cat-C (pink), N-cat (blue), and N-cat-C (green). The results indicate that the N- and C-terminal extentions alone affect the structure little, while an extended tail is formed when both of them are present.</p

Comparisons to structures of 2H family members.

<p>(a) Superposition of the CNPase/RICH structures. Colouring: mouse, light blue; human, green; rat, pink; goldfish RICH, gray. Note the conservation of the α7 helix in all structures; the 2′-AMP from the mouse CNPase active site is also shown. (b) Superposition of mouse CNPase (light blue) and human AKAP18 (orange), the two nucleotide-bound complexes of 2H family members. The C-terminus and the α7 helix of CNPase are labelled, and the covering C-terminal loop of AKAP18, present in non-CNPase 2H family members, is indicated by an arrow. The 2′-AMP in CNPase is in blue and the 5′-AMP in AKAP18 in magenta. Note that only the phosphate groups of the nucleotide ligands overlap.</p

Details of the active site.

<p>(a) An overall view of the CNPase active site. The apparent 2-fold symmetry axis of the active site is indicated by the green asterisk. The catalytic residues and 5 ordered water molecules are labelled. In this structure, the active site is occupied by a citrate molecule. (b) Stereo view of the binding mode of 2′-AMP to mouse CNPase. (c) Complex with NADP⁺. The double conformation of the region around Pro320 is also shown; the conformation in green corresponds to the one with the ligand, and the pink one to the conformation without bound ligand. (d) CNPase complex with two sulphate molecules. One sulphate resides in the active site (left) and the other binds to the α6-β5 loop (right).</p